JP2021055080A - Asymmetrically branched degradable polyethylene glycol derivative - Google Patents

Abstract

To provide a high-molecular-weight branched, degradable polyethylene glycol derivative which does not cause cell vacuoles.SOLUTION: A branched, degradable polyethylene glycol derivative represented by formula (1) includes an oligopeptide that decomposes intracellularly in each molecule. (In the formula, k1 and k2 are independently 1-12; j1 and j2 are independently 45-950; R is a hydrogen atom, a substituted or unsubstituted C1-12 alkyl group, a substituted aryl group, an aralkyl group, or a heteroalkyl group; Z is an oligopeptide which is degraded by intracellular enzymes; X is a functional group which can react with a biological substance; and L1 and L2 are each independently single bonds or divalent spacers).SELECTED DRAWING: None

Description

The present invention relates to a branched degradable polyethylene glycol derivative that decomposes in cells and is used for modifying biological substances.

Drugs using bio-related substances such as hormones, cytokines, antibodies, and enzymes are usually rapidly excreted from the body by glomerular filtration in the kidney and uptake by macrophages in the liver and spleen when administered into the body. It ends up. Therefore, the half-life in blood is short, and it is often difficult to obtain a sufficient pharmacological effect. In order to solve this problem, attempts have been made to chemically modify bio-related substances with hydrophilic polymers such as sugar chains and polyethylene glycol, albumin, and the like. As a result, it is possible to extend the half-life of biological substances in blood by increasing the molecular weight and forming a hydrated layer. It is also well known that modification with polyethylene glycol has effects such as reduction of toxicity and antigenicity of biorelated substances and improvement of solubility of poorly water-soluble drugs.

Bio-related substances modified with polyethylene glycol are covered with a hydration layer formed by ether bonds of polyethylene glycol and hydrogen bonds with water molecules, which increases the molecular size and thus avoids glomerular filtration in the kidneys. be able to. Furthermore, it is known that the interaction with opsonin and the cell surface constituting each tissue is reduced, and the transfer to each tissue is reduced. Polyethylene glycol is an excellent material that prolongs the half-life of biological substances in the blood, and it has been found that the higher the molecular weight, the higher the effect. So far, many studies have been conducted on bio-related substances modified with high molecular weight polyethylene glycol having a molecular weight of 40,000 or more, and the results have been obtained that the half-life in blood can be significantly extended.

Polyethylene glycol is regarded as the optimum standard among modifiers used for improving the performance of biological substances, and at present, a plurality of polyethylene glycol modified preparations have been put on the market and used in medical practice. On the other hand, when a bio-related substance modified with high molecular weight polyethylene glycol having a molecular weight of 40,000 or more was administered to an animal for a long period of time at a certain dose or more from the European Medicines Agency (EMA) in 2012, cells of some tissues A phenomenon in which vacuoles are generated inside has been reported (Non-Patent Document 1). At this time, there are no reports that the development of vacuoles itself has an adverse effect on the human body, and the dose used in the previous EMA report is extremely high compared to the dose generally applied in the medical field. Considering the high dose and the like, it can be said that there is no problem in the safety of the therapeutic preparations currently manufactured and sold and modified with polyethylene glycol having a molecular weight of 40,000 or more. However, in the treatment of very specific diseases (eg, dwarfism), it can be envisioned that a therapeutic protocol will be adopted in which the polyethylene glycol modified preparation is administered to the patient at a high dose for a long period of time. Therefore, it is expected that there is a potential demand for the development of polyethylene glycol-modified preparations that do not generate vacuoles in cells and can be applied even in such special situations.

In Non-Patent Document 2, when a large excess amount of polyethylene glycol was administered alone to an animal for a long period of time as compared with the usual dose of a polyethylene glycol modified preparation, no vacuole was observed at a molecular weight of 20,000, and the molecular weight was 40,000. Occurrence of vacuoles has been confirmed in. As one of the means for suppressing vacuoles, it is conceivable to reduce the molecular weight of polyethylene glycol, but if the molecular weight is reduced, there arises a problem that the half-life of bio-related substances in blood cannot be sufficiently improved.

There are reports on techniques for decomposing high molecular weight polyethylene glycol into low molecular weight polyethylene glycol in the body and promoting excretion from the kidneys. Patent Document 1 describes a polyethylene glycol derivative having a sulfide bond or a peptide bond site that is cleaved in a living body. There is a description that the polyethylene glycol derivative is decomposed in vivo to a molecular weight suitable for excretion from the kidney. However, no specific data on degradation have been shown, and there is no data that renal excretion was promoted. Furthermore, there is no description about cell vacuoles.

Patent Document 2 describes a polyethylene glycol derivative having an acetal moiety that can be hydrolyzed in a low pH environment in a living body. There is a description that the polyethylene glycol derivative is decomposed in vivo to a molecular weight suitable for excretion from the kidney. However, there is no specific data that the excretion from the kidney was promoted, and there is no description about cell vacuoles. Further, it is known that these acetal sites capable of hydrolysis are gradually decomposed even in blood, and it is expected that the half-life of the modified bio-related substance in blood cannot be sufficiently improved.

On the other hand, there are reports of polyethylene glycol derivatives into which degradable oligopeptides have been introduced in order to effectively release drugs, and hydrogels that decompose in the body.

Non-Patent Document 3 describes a polyethylene glycol derivative having an oligopeptide site that is degraded by an enzyme. Here, the oligopeptide is introduced as a linker between the anticancer agent and polyethylene glycol, and it has been reported that the oligopeptide is decomposed by an enzyme specifically expressed around the tumor to efficiently release the anticancer agent. The purpose is the release of anti-cancer agents, not the degradability of polyethylene glycol for the purpose of suppressing cell vacuoles.

Non-Patent Document 4 describes a hydrogel using a crosslinked molecule having an oligopeptide site that is decomposed by an enzyme and a multi-branched polyethylene glycol derivative. Here, the oligopeptide is used as a cross-linking molecule that connects multi-branched polyethylene glycol derivatives, and can further impart enzymatic degradability to the hydrogel. The purpose is to prepare a degradable hydrogel, not to impart degradability to polyethylene glycol for the purpose of suppressing cell vacuoles.

Patent Document 3 describes a branched polyethylene glycol derivative having an oligopeptide as a skeleton. Here, the oligopeptide is used as the basic skeleton of the polyethylene glycol derivative, and does not impart enzymatic degradability. In addition, oligopeptides are characterized by containing amino acids such as lysine and aspartic acid that have amino and carboxyl groups in their side chains, and the purpose is to synthesize branched polyethylene glycol derivatives that utilize them in the reaction. is there. It is not a polyethylene glycol derivative intended to suppress cell vacuoles.

Further, polyethylene glycol derivatives used for modifying bio-related substances generally have a linear type and a branched type, and in Non-Patent Document 5, the branched type is significantly more bio-related than the linear type. There is a description that it prolongs the half-life in blood. In recent years, most of the polyethylene glycol modified preparations on the market have adopted the branched type. However, there have been no reports on branched polyethylene glycol derivatives that suppress cell vacuoles in this field.

As described above, it is stable in blood, improves the half-life in blood of modified bio-related substances, and when taken up by cells, it specifically decomposes inside the cells and suppresses the development of vacuoles in the cells. There is a demand for a branched high-molecular-weight polyethylene glycol derivative that can be used.

Special Table 2009-527581 WO2005 / 108463 WO2006 / 08824

EMA / CHMP / SWP / 647258/2012 Daniel G. Rudmann, et al., Toxicol. Pathol., 41, 970-983 (2013) Francesco M Veronese, et al., Bioconjugate Chem., 16, 775-784 (2005) Jiyuan Yang, et al., Marcomol. Biosci., 10 (4), 445-454 (2010) Yulia Vugmeysterang, et al., Bioconjugate Chem., 23, 1452-1462 (2012)

An object of the present invention is to provide a high molecular weight branched polyethylene glycol derivative that does not cause cell vacuoles. More specifically, a branched degradable polyethylene glycol derivative that can be effectively used for modifying a biological substance, is stable in blood in the living body, and is decomposed in cells is industrially used. It is to be provided by a manufacturing method that can be produced in Japan.

As a result of diligent studies to solve the above problems, the present inventors have invented a branched degradable polyethylene glycol derivative having an oligopeptide that decomposes intracellularly.

That is, the present invention provides a branched degradable polyethylene glycol derivative represented by the following formula (1).
[1] A branched degradable polyethylene glycol derivative represented by the following formula (1), which has an oligopeptide that decomposes intracellularly in the molecule.

(In the formula, k ₁ and k ₂ are independently 1 to 12, j ₁ and j ₂ are independently 45 to 950, respectively, R is a hydrogen atom, and the number of carbon atoms is 1 which is substituted or not substituted. ~ 12 alkyl groups, substituted aryl groups, aralkyl groups or heteroalkyl groups, Z is an oligopeptide that is degraded by intracellular enzymes, X is a functional group that can react with biorelated substances, L ₁ and L ₂ is an independent single bond or divalent spacer.)

[2] The branched degradable polyethylene glycol derivative according to [1], wherein the degradable oligopeptide of Z is an oligopeptide having glycine as a C-terminal amino acid.

[3] The item according to any one of [1] to [2], wherein the degradable oligopeptide of Z is an oligopeptide having at least one hydrophobic neutral amino acid having a hydropathy index of 2.5 or more. Branched degradable polyethylene glycol derivative.

[4] The branched degradable polyethylene glycol derivative according to any one of [1] to [3], which has a total molecular weight of 20,000 or more.

[5] Even if L ₁ and L ₂ independently contain a single bond, a urethane bond, an amide bond, an ether bond, a thioether bond, a secondary amino group, a urea bond, or a bond and / or group thereof. The branched degradable polyethylene glycol derivative according to any one of [1] to [4], which is a good alkylene group.

[6] X is an active ester group, an active carbonate group, an aldehyde group, an isocyanate group, an isothiocyanate group, an epoxy group, a maleimide group, a vinylsulfonyl group, an acrylic group, a sulfonyloxy group, a carboxy group, a thiol group, a dithiopyridyl group, The branched type according to any one of [1] to [5], which is selected from the group consisting of an α-haloacetyl group, an alkynyl group, an allyl group, a vinyl group, an amino group, an oxyamino group, a hydrazide group and an azide group. Degradable polyethylene glycol derivative.

The present invention also provides, as another embodiment, a branched degradable polyethylene glycol derivative represented by the following formula (2).
[6] A branched degradable polyethylene glycol derivative represented by the following formula (2).

(In the formula, k ₁ and k ₂ are independently 1 to 12, j ₁ and j ₂ are independently 45 to 950, respectively, R is a hydrogen atom, and the number of carbon atoms is 1 which is substituted or not substituted. It is an alkyl group of ~ 4, a substituted aryl group, an aralkyl group or a heteroalkyl group, W is an oligopeptide of 5 to 47 residues having a symmetrical structure centered on glutamate or lysine, and a is 2 to 8. X is a functional group capable of reacting with a bio-related substance, and L ₁ and L ₂ are independently single-bonded or divalent spacers, respectively.)

[7] The branched degradable polyethylene glycol derivative according to [6], wherein the oligopeptide having a symmetrical structure centered on glutamic acid or lysine of W is an oligopeptide having the following w1 or w2 structure.

(In the formula, Q is a residue of glutamic acid or lysine, and Z is a degradable oligopeptide of 2 to 5 residues consisting of neutral amino acids excluding cysteine.)

[8] The degradable polyethylene glycol derivative according to [7], wherein the degradable oligopeptide of Z is an oligopeptide having glycine as a C-terminal amino acid.

[9] The item according to any one of [7] or [8], wherein the degradable oligopeptide of Z is an oligopeptide having at least one hydrophobic neutral amino acid having a hydropathy index of 2.5 or more. Branched degradable polyethylene glycol derivative.

[10] The branched degradable polyethylene glycol derivative according to any one of [6] to [9], which has a total molecular weight of 20,000 or more.

[11] Even if L ₁ and L ₂ independently contain a single bond, a urethane bond, an amide bond, an ether bond, a thioether bond, a secondary amino group, a urea bond, or a bond and / or group thereof. The branched degradable polyethylene glycol derivative according to any one of [6] to [10], which is a good alkylene group.

[12] X is an active ester group, an active carbonate group, an aldehyde group, an isocyanate group, an isothiocyanate group, an epoxy group, a maleimide group, a substituted maleimide group, a vinylsulfonyl group, an acrylic group, a substituted sulfonate group, a sulfonyloxy group and a carboxy group. , Mercapto group, pyridyldithio group, α-haloacetyl group, alkylcarbonyl group, iodoacetamide group, alkenyl group, alkynyl group, substituted alkynyl group, amino group, oxyamino group, hydrazide group and azide group. , The branched degradable polyethylene glycol derivative according to any one of [6] to [11].

The branched degradable polyethylene glycol derivative of the present invention is stable in blood in vivo and has an oligopeptide in its structure that is degraded by intracellular enzymes. Therefore, the branched degradable polyethylene glycol derivative is stable in blood and can impart a half-life in blood equivalent to that of a conventional non-degradable polyethylene glycol derivative to a biological substance. Furthermore, when the branched degradable polyethylene glycol derivative is taken up into cells, the oligopeptide site is rapidly degraded, so that it is possible to suppress the generation of cell vacuoles, which has been a problem until now. it can. Further, by limiting the oligopeptide to be introduced into polyethylene glycol to an oligopeptide having glycine as a C-terminal amino acid, impurities generated in the production process can be reduced and industrial production becomes possible.

Hereinafter, the present invention will be described in detail.
The branched degradable polyethylene glycol derivative according to the present invention is represented by the following formula (1).

In formula (1), k ₁ and k ₂ are independently 1 to 12, j ₁ and j ₂ are independently 45 to 950, respectively, and R is a hydrogen atom, substituted or unsubstituted carbon. The number 1 to 12 is an alkyl group, a substituted aryl group, an aralkyl group or a heteroalkyl group, Z is an oligopeptide that is decomposed by an intracellular enzyme, X is a functional group that can react with a bio-related substance, and L. ₁ and L ₂ are independently single-bonded or divalent spacers, respectively.

The total molecular weight of the polyethylene glycol derivative of the formula (1) of the present invention is usually 4,000 to 160,000, preferably 10,000 to 120,000, and more preferably 20,000 to 80,000. Is. In one preferred embodiment of the present invention, the polyethylene glycol derivative of the formula (1) of the present invention has a total molecular weight of 20,000 or more. The molecular weight referred to here is a number average molecular weight (Mn).

_{K 1} and k ₂ in the formula (1) are usually 1 to 12 independently of each other, preferably 1 to 6 independently of each other, and more preferably 1 to 2 independently of each other.

_{J 1} and j ₂ in the formula (1) are the number of repeating units of polyethylene glycol, respectively, and are usually 45 to 950 independently of each other, preferably 110 to 690 independently of each other, and more preferably each of them. Independently 220 to 480.

R in the formula (1) is a hydrogen atom, an alkyl group having 1 to 12 carbon atoms which is not substituted or substituted, a substituted aryl group, an aralkyl group or a heteroalkyl group. A "heteroalkyl group" is an alkyl group containing 1 to 5 heteroatoms selected from nitrogen, oxygen and sulfur atoms. R is preferably a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, more preferably a hydrogen atom, a methyl group or an ethyl group, and further preferably a hydrogen atom.

_{L 1} and L ₂ in the formula (1) are independently single-bonded or divalent spacers, respectively, but these spacers are not particularly limited as long as they are groups capable of forming a covalent bond, but are preferable. Is a phenylene group, an amide bond, an ether bond, a thioether bond, a urethane bond, a secondary amino group, a carbonyl group, a urea bond, or an alkylene group which may contain these bonds and / or a group, more preferably. , An alkylene group, an amide bond, an ether bond, a urethane bond, a secondary amino group, or a group formed by bonding a carbonyl group and an alkylene group, and particularly preferable embodiments are shown in the following group (I). It is a thing. Further, 2 to 5 spacers of the group (I) may be combined. Ester bonds and carbonate bonds as divalent spacers are not suitable because they gradually decompose in the blood in the living body.

Group (I):

In (z1) to (z11), s in the equation indicates an integer of 0 to 10, preferably an integer of 0 to 6, and more preferably an integer of 0 to 3. Further, in (z2) to (z11), s in the equation may be the same or different.

_{L 1} in the formula (1) is a single bond or group (I) (z2), (z3), (z4), (z6), (z7), (z8), (z9), (z10), The combination of (z2) and (z6) is preferable, and the combination of single bond, (z3), (z6), (z9), (z10), (z2) and (z6) is more preferable.
_{L 2} in the formula (1) is (z1), (z2), (z3), (z4), (z5), (z6), (z7), (z8), (z11) of the group (I). Is preferable, and (z3), (z5), and (z11) are more preferable.

Z in the formula (1) is not particularly limited as long as it is an oligopeptide that is stable in the blood in the living body and decomposed by an intracellular enzyme, but is 2 to 8 residues consisting of neutral amino acids excluding cysteine. The oligopeptide of the group is preferable, an oligopeptide of 2 to 6 residues consisting of a neutral amino acid excluding cysteine is more preferable, and an oligopeptide of 2 to 4 residues consisting of a neutral amino acid excluding cysteine is further preferable.

Further, Z in the formula (1) may be an oligopeptide composed of an amino acid having an amino group or a carboxyl group in the side chain, specifically, a neutral amino acid containing no lysine, aspartic acid or glutamic acid. preferable. The amino acid used here is an α-amino acid and is basically L-type.

Furthermore, since cysteine, which is a neutral amino acid, has a thiol group and forms a disulfide bond with other thiol groups, Z in the formula (1) is an oligopeptide composed of a neutral amino acid containing no cysteine. It is desirable to have.

In addition, Z in the formula (1) is preferably an oligopeptide having glycine as a C-terminal amino acid. When reacting the C-terminal carboxyl group with the polyethylene glycol derivative, it is basically necessary to activate the C-terminal carboxyl group with a condensing agent or the like. It is known that in this activation step, amino acids other than glycine are prone to epimerization, and stereoisomers are by-produced. By using achiral glycine as the C-terminal amino acid of the oligopeptide, a high-purity target product without by-products of the stereoisomer can be obtained.

Further, Z in the formula (1) is an oligopeptide having at least one hydrophobic neutral amino acid having a hydropathy index of 2.5 or more, specifically, phenylalanine, leucine, valine, and isoleucine. Is preferable, and an oligopeptide having phenylalanine is more preferable. The hydropathy index, which was created by Kyte and Doolittle to quantitatively indicate the hydrophobicity of amino acids, indicates that the larger the value, the more hydrophobic the amino acid (Kyte J & Doolittle RF, 1982, J Mol). Biol, 157: 105-132.).

Z in the formula (1) may be an oligopeptide having 2 to 8 residues consisting of neutral amino acids excluding cysteine, which is stable in the blood in the living body and has the ability to be decomposed by intracellular enzymes. There are no particular restrictions, but specific examples include glycine-phenylalanine-leucine-glycine, glycine-glycine-phenylalanine-glycine, glycine-phenylalanine-glycine, glycine-leucine-glycine, valine-citrulin-glycine, valine-. Alanin-glycine, glycine-glycine-glycine, phenylalanine-glycine, etc., preferably glycine-phenylalanine-leucine-glycine, glycine-glycine-phenylalanine-glycine, glycine-phenylalanine-glycine, glycine-glycine-glycine, valine-citrulin. -Glycine, valine-alanine-glycine, phenylalanine-glycine, more preferably glycine-phenylalanine-leucine-glycine, glycine-phenylalanine-glycine, valine-citrulin-glycine, valine-alanine-glycine, even more preferably. Glycine-phenylalanine-leucine-glycine, valine-citrulin-glycine.

X in the formula (1) is particularly limited as long as it is a functional group that forms a covalent bond by reacting with a functional group existing in a biorelated substance such as a physiologically active protein, peptide, antibody, or nucleic acid to be chemically modified. Not done. For example, "Harris, JM Poly (Ethylene Glycol) Chemistry; Plenum Press: New York, 1992", "Hermanson, GT Bioconjugate Techniques, 2nd ed .; Academic Press: San Diego, CA, 2008" and "PEGylated Protein Drugs: Basic" Science and Clinical Applications; Veronese, FM, Ed .; Birkhauser: Basel, Switzerland, 2009 ”etc.

The "functional group capable of reacting with a bio-related substance" represented by X in the formula (1) is a functional group such as an amino group, a mercapto group, an aldehyde group, a carboxy group, an unsaturated bond or an azido group possessed by the bio-related substance. It is not particularly limited as long as it is a functional group capable of chemically bonding with.
Specifically, an active ester group, an active carbonate group, an aldehyde group, an isocyanate group, an isothiocyanate group, an epoxy group, a carboxy group, a mercapto group, a maleimide group, a substituted maleimide group, a hydrazide group, a dithiopyridyl group, a substituted sulfonate group, vinylsulfonyl group, an amino group, an oxy amino group (H 2 _N-O-group), iodoacetamide group, an alkylcarbonyl group, an alkenyl group (e.g., allyl group, vinyl group), alkynyl group, substituted alkynyl group (e.g., below (Alkinyl group substituted with a hydrocarbon group having 1 to 5 carbon atoms), an azide group, an acrylic group, a sulfonyloxy group (for example, an alkylsulfonyloxy group), an α-haloacetyl group and the like, preferably an active ester. Group, active carbonate group, aldehyde group, isocyanate group, isothiocyanate group, epoxy group, maleimide group, substituted maleimide group, vinylsulfonyl group, acrylic group, sulfonyloxy group (for example, alkyl-sulfonyloxy group having 1 to 5 carbon atoms). ), Substituted sulfonate group, carboxy group, mercapto group, pyridyldithio group, α-haloacetyl group, alkynyl group, substituted alkynyl group (for example, 2 to 5 carbon atoms substituted with the hydrocarbon group having 1 to 5 carbon atoms described later). Alkinyl group), allyl group, vinyl group, amino group, oxyamino group, hydrazide group and azide group, more preferably active ester group, active carbonate group, aldehyde group, maleimide group, oxyamino group and amino group. Yes, particularly preferably an active ester group, an active carbonate group, an aldehyde group, a maleimide group and an oxyamino group.

In another preferred embodiment, such functional groups X can be classified into the following groups (II), group (III), group (IV), group (V), group (VI) and group (VII). it can.

Group (II): Functional groups capable of reacting with amino groups of bio-related substances The following (a), (b), (c), (d), (e), (f), (g), (j) ), Or the group represented by (k).

Group (III): Functional groups capable of reacting with mercapto groups of bio-related substances The following (a), (b), (c), (d), (e), (f), (g), (h) ), (I), (j), (k), or (l).

Group (IV): Functional groups capable of reacting with aldehyde groups contained in biorelated substances Examples thereof include groups represented by (h), (m), (n), or (p) below.

Group (V): Functional group capable of reacting with a carboxyl group of a bio-related substance Examples thereof include groups represented by (h), (m), (n), or (p) below.

Group (VI): Functional groups capable of reacting with unsaturated bonds possessed by biorelated substances Examples thereof include groups represented by (h), (m), or (o) below.

Group (VII): Functional groups capable of reacting with azide groups contained in bio-related substances Examples thereof include the groups represented by (l) below.

In the functional group (j), W ₁ in the formula represents a halogen atom such as a chlorine atom (Cl), a bromine atom (Br) or an iodine atom (I), preferably Br, or I, more preferably I. ..

Further, in the functional group (e) and the functional group (l), Y ¹ and Y ³ in the formula independently represent a hydrogen atom or a hydrocarbon group having 1 to 5 carbon atoms, preferably 1 carbon atom. ~ 5 hydrocarbon groups. Specific examples of the hydrocarbon group having 1 to 5 carbon atoms include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, a tertiary butyl group and the like, and a methyl group or an ethyl group is preferable. is there.

Further, in the functional group (k), Y ² in the formula represents a hydrocarbon group having 1 to 10 carbon atoms which may contain a fluorine atom, and specifically, a methyl group, an ethyl group, a propyl group, and the like. Isopropyl group, butyl group, tertiary butyl group, hexyl group, nonyl group, vinyl group, phenyl group, benzyl group, 4-methylphenyl group, trifluoromethyl group, 2,2,2-trifluoroethyl group, 4- Examples thereof include a (trifluoromethoxy) phenyl group, preferably a methyl group, a vinyl group, a 4-methylphenyl group, or a 2,2,2-trifluoroethyl group.

The active ester group is an ester group having an alkoxy group having a high desorption ability. Examples of the alkoxy group having high desorption ability include an alkoxy group derived from nitrophenol, N-hydroxysuccinimide, pentafluorophenol and the like. The active ester group is preferably an ester group having an alkoxy group derived from N-hydroxysuccinimide.

The active carbonate group is a carbonate group having an alkoxy group having a high desorption ability. Examples of the alkoxy group having high desorption ability include an alkoxy group derived from nitrophenol, N-hydroxysuccinimide, pentafluorophenol and the like. The active carbonate group is preferably a carbonate group having an alkoxy group derived from nitrophenol or N-hydroxysuccinimide.

The substituted maleimide group is a maleimide group in which a hydrocarbon group is bonded to one carbon atom of the double bond of the maleimide group. Specific examples of the hydrocarbon group include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, a tertiary butyl group and the like, and a methyl group or an ethyl group is preferable.

The substituted sulfonate group is a sulfonate group in which a hydrocarbon group which may contain a fluorine atom is bonded to a sulfur atom of the sulfonate group. Specific examples of the hydrocarbon group that may contain a fluorine atom include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, a tertiary butyl group, a hexyl group, a nonyl group, a vinyl group, and a phenyl group. , Benzyl group, 4-methylphenyl group, trifluoromethyl group, 2,2,2-trifluoroethyl group, 4- (trifluoromethoxy) phenyl group and the like, preferably methyl group, vinyl group, 4- It is a methylphenyl group or a 2,2,2-trifluoroethyl group.

Preferable examples of the branched degradable polyethylene glycol derivative of the formula (1) of the present invention include the following branched degradable polyethylene glycol derivatives.
[Branched degradable polyethylene glycol derivative (1-1)]
k ₁ and k ₂ are 1 to 2 independently of each other;
j ₁ and j ₂ are 220-480, respectively;
R is a hydrogen atom;
Z is a 2-8 residue oligopeptide consisting of neutral amino acids excluding cysteine (eg, phenylalanine-glycine);
X is an active ester group (eg, N-succinimidyloxycarbonyl group), an aldehyde group (eg, N- (formylethyl) carbamoyl group), a carboxyl group, a maleimide group (eg, N- (N-maleimi)). Selected from the group consisting of a zirethylcarbonylaminoethyl) carbamoyl group) and an amino group (eg, N- (aminoethyl) carbamoyl group);
L ₁ and L ₂ are alkylene groups (eg, propylene groups) that may independently contain a secondary amino group and / or a carbonyl group;
A branched degradable polyethylene glycol derivative of the formula (1).

The branched degradable polyethylene glycol derivative represented by the formula (1) can be produced, for example, by the following steps.

Reaction 1

A in Reaction 1 is a leaving group, and R, j ₁ , k ₁ , and k ₂ are synonymous with the above.

A in the formula (3) is a leaving group, and is not particularly limited as long as it is a leaving group having a reactivity in the coupling reaction. For example, a chloro group, a bromo group, an iodo group, a mesylate group, and a tosylate group. , Chloromethanesulfonate group and the like.

In reaction 1, the polyethylene glycol derivative represented by the formula (3) and the compound represented by the formula (4) are coupled in an anhydrous solvent in the presence of a strong base catalyst, and the polyethylene represented by the formula (5) is reacted. This is a step of obtaining a glycol derivative.

The strong base catalyst in the coupling reaction is not particularly limited as long as it is a strong base catalyst in which the reaction proceeds, and examples thereof include potassium hydroxide, sodium hydroxide, sodium methoxide, and sodium ethoxide.
The anhydrous solvent in the coupling reaction is not particularly limited as long as it is a solvent that does not react with the compounds represented by the formulas (3) and (4), and for example, tetrahydrofuran, acetonitrile, DMF, dichloromethane, chloroform and the like can be used. Examples include aprotic polar solvents and mixtures thereof.
It is preferable to purify and remove impurities produced as a by-product in the reaction, compounds remaining unconsumed in the reaction, and a strong base catalyst. Purification is not particularly limited, but purification can be performed by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction and the like.

Reaction 2

Pro in Reaction 2 is a protecting group, Peptide is an oligopeptide, L ₃ is a single bond or divalent spacer synonymous with _{L 1} and L _{2 , and j 2} is synonymous with the above.

Pro in Reaction 2 is a protecting group, where the protecting group is a component that prevents or prevents the reaction of a particular chemically reactive functional group in the molecule under certain reaction conditions. Protecting groups vary depending on the type of chemically reactive functional group protected, the conditions used and the presence of other functional or protecting groups in the molecule. Specific examples of protecting groups can be found in many common books, such as "Wuts, PGM; Greene, TW Protective Groups in Organic Synthesis, 4th ed .; Wiley-Interscience: New York, 2007". It is described in. Further, the functional group protected by the protecting group can be regenerated by deprotecting, that is, chemically reacting with the reaction conditions suitable for each protecting group. Typical deprotection conditions for protecting groups are described in the aforementioned literature.

In Reaction 2, the carboxyl group of the oligopeptide in which the N-terminal amino group represented by the formula (6) is protected by a protecting group and the polyethylene glycol derivative represented by the formula (7) having a methoxy group at one end are used. This is a step of bonding amino groups by a condensation reaction to obtain a polyethylene glycol derivative represented by the formula (8).

The protecting group for the N-terminal amino group of the oligopeptide is not particularly limited, and examples thereof include an acyl-based protecting group and a carbamate-based protecting group, specifically, a trifluoroacetyl group and 9-fluorenylmethyloxycarbonyl. Examples include a group (Fmoc), a tert-butyloxycarbonyl group and the like.

The condensation reaction is not particularly limited, but a reaction using a condensing agent is desirable. As the condensing agent, a carbodiimide-based condensing agent such as dicyclohexylcarbodiimide (DCC) or 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) may be used alone, or N-hydroxysuccinimide may be used alone. It may be used in combination with a reagent such as (NHS), 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt). In addition, more reactive HATU, HBTU, TATU, TBTU, COMU, 4- (4,6-dimethoxy-1,3,5-triazine-2-yl) -4-methylmorpholinium chloride n hydrate A condensing agent such as (DMT-MM) may be used. Further, in order to promote the reaction, a base such as triethylamine or dimethylaminopyridine may be used.

Impurities produced as a by-product in the reaction, or oligopeptides and condensing agents remaining unconsumed in the reaction are preferably purified and removed. Purification is not particularly limited, but can be purified by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction and the like.

Deprotection 3

Peptide, L ₃ and j ₂ are synonymous with the above.

The deprotection 3 is a step of deprotecting the protecting group of the polyethylene glycol derivative represented by the formula (8) obtained in the reaction 2 to obtain the polyethylene glycol derivative represented by the formula (9). The deprotection reaction can be a conventionally known method, it is necessary to use the conditions divalent spacer oligopeptide or L ₃ is not decomposed. Further, this step can also be carried out as a part of the step of reaction 2.

Impurities produced by the deprotection reaction are preferably purified and removed. Purification is not particularly limited, but can be purified by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction and the like.

Reaction 4

_{K 3 in} reaction 4 is an integer of 1 to 6, and Peptide, j ₂ and L ₃ are synonymous with the above.

_{K 3} in the formula (8) is an integer of 1 to 6, preferably an integer of 2 to 4.

Reaction 4 is a step of reacting the amino group at the terminal of the polyethylene glycol derivative represented by the formula (9) obtained by deprotection 3 with the compound represented by the formula (10) in the presence of a base catalyst to convert it into a carboxyl group. Is.

Impurities and the like produced as a by-product in Reaction 4 are preferably purified and removed. Purification is not particularly limited, but can be purified by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction and the like.

Reaction 5

Peptide, L ₃ , k ₃ , and j _{2 in} Reaction 5 are synonymous with the above.

In the reaction 5, the polyethylene glycol derivative represented by the formula (11) obtained in the reaction 4 was reacted with the compound represented by the formula (12) in the presence of a base catalyst, and the active ester group was introduced into the reaction 5 according to the formula (13). It is a step of obtaining the polyethylene glycol derivative represented, and this step can also be carried out as a part of the step of Reaction 4.

Reaction 6

Peptide, R, L ₃ , j ₁ , j ₂ , k ₁ , k ₂ , and k ₃ in Reaction 6 are synonymous with the above.

In Reaction 6, the amino group of the polyethylene glycol derivative represented by the formula (5) obtained in Reaction 1 and the active ester group of the polyethylene glycol derivative represented by the formula (13) obtained in Reaction 5 are bonded by the reaction. , A step of obtaining a branched degradable polyethylene glycol derivative represented by the formula (14).

Impurities produced as a by-product in the reaction or polyethylene glycol derivatives remaining unconsumed in the reaction are preferably purified and removed. Purification is not particularly limited, but purification can be performed by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction and the like.

A typical example of the step of converting the terminal carboxyl group of the polyethylene glycol derivative represented by the formula (14) into another functional group will be described below, but the conversion method is not limited thereto.

For example, when it is desired to convert the terminal carboxyl group of the polyethylene glycol derivative represented by the formula (15) into a maleimide group, the desired product can be obtained by subjecting it to a condensation reaction with N- (2-aminoethyl) maleimide in the presence of a base catalyst. Obtainable.

For example, when it is desired to convert the terminal carboxyl group of the polyethylene glycol derivative represented by the formula (14) into an amino group, N- (9-H-fluorolen-9-ylmethoxycarbonyl) -1,2-ethanediamine and a base It can be obtained by conducting a condensation reaction in the presence of a catalyst and then performing a deprotection reaction.

Since these reaction reagents are low molecular weight reagents and have significantly different solubility from polyethylene glycol derivatives which are high molecular weight polymers, they can be easily removed by a general purification method such as extraction or crystallization.

As still another aspect of the present invention, a branched degradable polyethylene glycol derivative represented by the following formula (2) is provided.

In formula (2), k ₁ and k ₂ are independently 1 to 12, j ₁ and j ₂ are independently 45 to 950, respectively, and R is a hydrogen atom, substituted or unsubstituted carbon. It is an alkyl group of number 1 to 4, a substituted aryl group, an aralkyl group or a heteroalkyl group, W is an oligopeptide having a symmetric structure centered on glutamate or lysine, and a is an oligopeptide of 5 to 47 residues, and a is 2 to 8. Yes, X is a functional group capable of reacting with a bio-related substance, and L ₁ and L ₂ are independently single-bonded or divalent spacers, respectively.

The total molecular weight of the polyethylene glycol derivative of the formula (2) of the present invention is usually 4,000 to 160,000, preferably 10,000 to 120,000, and more preferably 20,000 to 80,000. Is. In one preferred embodiment of the present invention, the polyethylene glycol derivative of the formula (2) of the present invention has a total molecular weight of 20,000 or more. The molecular weight referred to here is a number average molecular weight (Mn).

_{K 1} and k ₂ in the formula (2) are usually 1 to 12 independently of each other, preferably 1 to 6 independently of each other, and more preferably 1 to 2 independently of each other.

_{J 1} and j ₂ in the formula (2) are the number of repeating units of polyethylene glycol, respectively, and are usually 45 to 950 independently of each other, preferably 110 to 690 independently of each other, and more preferably each of them. Independently 220 to 480.

W in the formula (2) is an oligopeptide having a symmetrical structure centered on glutamic acid or lysine, with 5 to 47 residues, preferably 5 to 27 residues, more preferably 5 to 19 residues, and is in vivo. There is no particular limitation as long as it is an oligopeptide that is stable in blood and decomposed by intracellular enzymes, but the amino acids that make up the oligopeptide are those other than glutamic acid or lysine that make up the central part, except for cysteine. It preferably consists of sex amino acids. The oligopeptide having a symmetrical structure centered on glutamic acid or lysine is a peptide having the same peptide as the α-position carboxyl group and γ-position carboxyl group of glutamic acid or the α-position amino group and ε-position amino group of lysine. It means a bound compound, and is an oligopeptide in which a pair of peptides centered on glutamate or lysine has a symmetrical structure. The composition ratio (number of neutral amino acids / number of glutamic acid) between the number of neutral amino acids and the number of glutamic acid or lysine in the oligopeptide is usually 2 to 10, preferably 2 to 8, and even more preferably. 2 to 6 The amino acids that make up W are basically L-type.

Particularly preferred embodiments of W are those shown in the group (VIII) below.
Group (VIII):

(In the formula, Q is a residue of glutamic acid or lysine, and Z is a degradable oligopeptide of 2 to 5 residues consisting of neutral amino acids excluding cysteine.)

A in the formula (2) is the number of polyethylene glycol chains bound to the oligopeptide represented by W, which is usually 2 to 8, preferably 2 or 4 or 8, and more preferably 2. Or 4.

The preferred embodiments of R, X, L ₁ , L ₂ and Z in (w1) to (w2) are as described in the above formula (1).

One of the preferred embodiments of the formula (2) is a tri-branched degradable polyethylene glycol derivative represented by the following formula (15) in which W is w1 and a = 2.

(In the formula, Q, Z, R, k ₁ , k ₂ , j ₁ , j ₂ , X, L ₁ and L ₂ are synonymous with the above.)

One of the preferred embodiments of the formula (2) is a 5-branched degradable polyethylene glycol derivative represented by the following formula (16) in which W is w2 and a = 4.

(In the formula, Q, R, Z, k ₁ , k ₂ , j ₁ , j ₂ , X, L ₁ and L ₂ are synonymous with the above.)

Preferable examples of the branched degradable polyethylene glycol derivative of the formula (2) of the present invention include the following branched degradable polyethylene glycol derivatives.
[Branched degradable polyethylene glycol derivative (2-1)]
k ₁ and k ₂ are 1 to 2 independently of each other;
j ₁ and j ₂ are 220-480, respectively;
R is a hydrogen atom;
W is a 5-9 residue oligopeptide having a symmetrical structure centered on glutamic acid or lysine (eg, glycine-phenylalanine-glutamic acid-phenylalanine-glycine);
a is 2 or 4;
X is the carboxyl group;
L ₁ and L ₂ are alkylene groups (eg, propylene group, pentylene group) which may independently contain an ether bond, a secondary amino group and / or a carbonyl group;
A branched degradable polyethylene glycol derivative of the formula (2).

The branched degradable polyethylene glycol derivative of the present invention can be produced, for example, by the following steps when Q is a residue of glutamic acid.

Reaction 7

Pro, Peptide, L ₃ and j _{2 in} Reaction 7 are synonymous with the above.

Reaction 7 has two carboxyls, an amino group of the polyethylene glycol derivative represented by the formula (9) obtained by deprotection 3 and a glutamate derivative in which the amino group represented by the formula (17) is protected by a protecting group. This is a step of bonding groups by a condensation reaction to obtain a branched polyethylene glycol derivative represented by the formula (18), which has a structure in which two degradable polyethylene glycol chains are linked by glutamate residues.
Similar to the above reaction 2, a reaction using a condensing agent is desirable, and in order to accelerate the reaction, a base such as triethylamine or dimethylaminopyridine may be used.
The protecting group for the amino group of glutamic acid is not particularly limited, and examples thereof include an acyl-based protecting group and a carbamate-based protecting group, specifically, a trifluoroacetyl group, a 9-fluorenylmethyloxycarbonyl group and a t-. Examples include a butyloxycarbonyl group.
Impurities produced as a by-product in the reaction or polyethylene glycol derivatives remaining unconsumed in the reaction are preferably purified and removed. Purification is not particularly limited, but can be purified by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction and the like.

Deprotection 8

Peptide in deprotection 8, _{L 3} and _{j 2} are as defined above.

The deprotection 8 is a step of deprotecting the protecting group of the polyethylene glycol derivative represented by the formula (18) obtained in the reaction 7 to obtain the polyethylene glycol derivative represented by the formula (19). The reaction and purification are possible under the same conditions as the deprotection 3.
As a method for removing polyethylene glycol impurities having different molecular weights and functional groups from the polyethylene glycol derivative represented by the formula (19), the purification techniques described in JP-A-2014-208786 and JP-A-2011-79934 are used. Can be done.

Reaction 9

Pro, Peptide, L ₃ and j _{2 in} Reaction 9 are synonymous with the above.

In the reaction 9, the amino group of the polyethylene glycol derivative represented by the formula (19) obtained by deprotection 8 and the glutamate derivative in which the amino group represented by the formula (17) is protected by a protecting group are two carboxyls. This is a step of bonding groups by a condensation reaction to obtain a branched polyethylene glycol derivative represented by the formula (20), which has a structure in which four degradable polyethylene glycol chains are linked by glutamate residues. The reaction and purification are possible under the same conditions as in Reaction 2.

Deprotection 10

Peptide in the deprotection 10, _{L 3} and _{j 2} are as defined above.

The deprotection 10 is a step of deprotecting the protecting group of the polyethylene glycol derivative represented by the formula (20) obtained in the reaction 9 to obtain the polyethylene glycol derivative represented by the formula (21). The reaction and purification are possible under the same conditions as the deprotection 8. Further, this step can also be carried out in a series of steps of reaction 9.

Reaction 11

Peptide, L ₃ , j ₂ and k _{3 in} Reaction 11 are synonymous with the above.

In the reaction 11, the amino group at the end of the bifurcated polyethylene glycol derivative represented by the formula (19) obtained by deprotection 8 is reacted with the compound represented by the formula (22) in the presence of a base catalyst to form a hydroxyl group. This is a step of converting to obtain a bifurcated polyethylene glycol derivative represented by the formula (23). The reaction and purification are possible under the same conditions as in Reaction 4.

Further, instead of the bifurcated polyethylene glycol derivative represented by the formula (19) in the reaction 11, a tetrabranched polyethylene glycol derivative represented by the formula (21) obtained by deprotection 10 is used as a raw material. As a result, a 4-branched polyethylene glycol derivative represented by the following formula (24) can be obtained.

_{Peptide, L 3} , j ₂ and k ₃ of the formula (24) are synonymous with the above.

Reaction 12

Peptide, L ₃ , j ₂ and k _{3 in} Reaction 12 are synonymous with the above.

In the reaction 12, the hydroxyl group of the bifurcated polyethylene glycol derivative represented by the formula (23) obtained in the reaction 11 was reacted with the compound represented by the formula (25), and the active carbonate group was introduced into the reaction 12 (26). It is a step of obtaining the bifurcated polyethylene glycol derivative represented by the above, and this step can be carried out as a part of the affirmation of the reaction 11, and the reaction and purification can be carried out under the same conditions as the reaction 5.

Further, by using a 4-branched polyethylene glycol derivative represented by the formula (24) as a raw material instead of the bi-branched polyethylene glycol derivative represented by the formula (23) in the reaction 12, the following formula (27) is used. ) Can be obtained as a tetrabranched polyethylene glycol derivative.

_{Peptide, L 3} , j ₂ and k ₃ of the formula (27) are synonymous with the above.

Reaction 13

Peptide, R, L ₃ , j ₁ , j ₂ , k ₁ , k ₂ and k ₃ in Reaction 13 are synonymous with the above.

In Reaction 13, the amino group of the polyethylene glycol derivative represented by the formula (5) obtained in Reaction 1 and the active ester group of the polyethylene glycol derivative represented by the formula (26) obtained in Reaction 12 are bonded by the reaction. , A step of obtaining a tri-branched degradable polyethylene glycol derivative represented by the formula (28), and the reaction and purification can be carried out under the same conditions as in the reaction 6.

Further, by using a 4-branched polyethylene glycol derivative represented by the formula (27) as a raw material instead of the bi-branched polyethylene glycol derivative represented by the formula (26) in the reaction 13, the following formula (29) is used. ) Can be obtained as a 5-branched polyethylene glycol derivative.

Peptide, R, L ₃ , j ₁ , j ₂ , k ₁ , k ₂ and k ₃ in formula (29) are synonymous with the above.

A typical example of the step of converting the terminal carboxyl group of the branched polyethylene glycol derivative represented by the formula (28) or the formula (29) to another functional group will be described below. It is not limited.

For example, when it is desired to convert the terminal carboxyl group of the branched polyethylene glycol derivative represented by the formula (28) or the formula (29) into a maleimide group, it is condensed with N- (2-aminoethyl) maleimide in the presence of a base catalyst. By reacting, the desired product can be obtained.

For example, when it is desired to convert the terminal carboxyl group of the polyethylene glycol derivative represented by the formula (28) or the formula (29) into an amino group, N- (9-H-fluorolen-9-ylmethoxycarbonyl) -1, 2 -It can be obtained by conducting a condensation reaction with ethanediamine in the presence of a base catalyst and then performing a deprotection reaction.

Since these reaction reagents are low molecular weight reagents and have significantly different solubility from polyethylene glycol derivatives which are high molecular weight polymers, they can be easily removed by general purification methods such as extraction and crystallization. ..

The branched degradable polyethylene glycol obtained through the above steps is required to be stable in blood and have the ability to decompose only intracellularly. In order to appropriately evaluate its performance, for example, the following tests can be carried out to evaluate the stability of branched degradable polyethylene glycol in blood and its degradability in cells.
In consideration of the influence of the type of functional group of the polyethylene glycol derivative in these evaluations, all the evaluation samples were unified to the polyethylene glycol derivative having one amino group.

The test method for evaluating the stability of the branched degradable polyethylene glycol derivative in blood is not particularly limited, and examples thereof include tests using sera of mice, rats, humans and the like. Specifically, the polyethylene glycol derivative is dissolved in serum to a concentration of 1 to 10 mg / mL, incubated at 37 ° C. for 96 hours, and then the polyethylene glycol derivative contained in the serum is recovered and GPC is measured. The decomposition rate can be evaluated with. The decomposition rate is calculated from the peak area% of the GPC membrane of the polyethylene glycol derivative before the stability test and the peak area% of the GPC membrane of the polyethylene glycol derivative after the stability test. Specifically, the following formula is used.
Decomposition rate = (Peak area% before test-Peak area% after test) ÷ Peak area% before test x 100
For example, if the peak area% of the GPC membrane of the branched degradable polyethylene glycol derivative before the stability test is 95% and the peak area% of the GPC membrane after the test is 90%, the decomposition rate is as follows. It is calculated as.
Decomposition rate = (95-90) ÷ 95 × 100 = 5.26 (%)
If the branched degradable polyethylene glycol derivative is decomposed in blood, the desired half-life in blood cannot be obtained. Therefore, in the stability test, the decomposition rate after 96 hours is 10% or less. It is preferably 5% or less, more preferably 5% or less.

The test method for evaluating the intracellular degradability of the branched degradable polyethylene glycol derivative is not particularly limited, but for example, a medium containing the branched degradable polyethylene glycol derivative is used in the cells. Examples include a test for culturing. The cells and medium used here are not particularly limited, but specifically, a polyethylene glycol derivative is dissolved in RPMI-1640, which is a medium, so as to have a concentration of 1 to 20 mg / mL, and this medium is used. After culturing the macrophage cell RAW264.7 at 37 ° C. for 96 hours, the polyethylene glycol derivative in the cell is recovered, and the degradation rate can be evaluated by measuring GPC. The decomposition rate is calculated by using the peak area% of the GPC membrane of the polyethylene glycol derivative before and after the test, as in the stability test.
For example, suppose that the peak area% of the GPC membrane of the branched degradable polyethylene glycol derivative before the degradability test using cells was 95%, and the peak area% of the GPC membrane after the test was 5%. The decomposition rate is calculated as follows.
Decomposition rate = (95-5) ÷ 95 × 100 = 94.7 (%)
Since the branched degradable polyethylene glycol derivative cannot suppress the vacuoles of the target cells unless it is efficiently degraded inside the cells, the degradation rate after 96 hours is preferably 90% or more in the degradation test. 95% or more is more preferable.

Non-Patent Document 2 describes that the vacuolation of cells by high molecular weight polyethylene glycol is related to the accumulation of polyethylene glycol in tissues. The test method for evaluating the accumulation of the degradable polyethylene glycol derivative in cells is not particularly limited, but can be evaluated based on the above-mentioned intracellular degradability.

Hereinafter, the present invention will be described in more detail based on Examples, but the present invention is not limited to the following Examples.

^{The 1} H-NMR obtained in the following examples was obtained from JNM-ECP400 or JNM-ECA600 manufactured by JEOL Datum Co., Ltd. For the measurement using the φ5mm tube, the deuterated solvent, using CDCl ₃ and _d 6-DMSO containing tetramethylsilane (TMS) as _{D 2} O, or internal standard. The molecular weight and terminal functional group purity of the obtained polyethylene glycol derivative were calculated using liquid chromatography (GPC and HPLC). As the liquid chromatography system, "HLC-8320GPC EcoSEC" manufactured by Tosoh Corporation was used for GPC, and "ALLIANCE" manufactured by WATERS Co., Ltd. was used for HPLC.

[Example 1]

[Example 1-1]

Average molecular weight = 20,000, "SUNBRIGHT MEH-20T" (manufactured by Nichiyu Co., Ltd.) (10 g) was dissolved in toluene (40 g), dehydrated by reflux at 110 ° C. for 1 hour, cooled to 40 ° C., and triethylamine (80 mg). ), Methanesulfonyl chloride (84 mg) was added, and the mixture was reacted at 40 ° C. for 3 hours. After completion of the reaction, the mixture was diluted with toluene (100 g), hexane (100 g) was added, and the mixture was stirred at room temperature for 30 minutes to precipitate the product. The precipitate was collected by suction filtration using a 5A filter paper, dissolved in ethyl acetate (200 g), hexane (100 g) was added, and the mixture was stirred at room temperature for 15 minutes to precipitate the product. The precipitate was collected by suction filtration using a 5A filter paper, washed with hexane (100 g), suction filtered using a 5A filter paper, and vacuum dried to obtain compound (p1). Yield 8.9g.
_{^{1 H-NMR (CDCl 3)}} : 3.08ppm (s, 3H, -O-SO 2 - CH 3) 3.38ppm (s, 3H, -O-CH 2 -CH 2 - (O-CH 2 -CH _{2) j -O- CH 3),} 3.64ppm (m, about _{1,900H, -O-CH 2 -CH 2} - (O- CH 2 -CH 2) j -O-CH 3)

[Example 1-2]

Glycine hydrochloride (5 g) was dissolved in ion-exchanged water (50 g). NaOH (3.0 g) was added to the aqueous glycine solution to adjust the pH to 10.8. Then, the compound (p1) (8 g) obtained in Example 1-1 was added to the aqueous solution, and the solution was reacted at 40 ° C. for 72 hours. After the reaction, the pH of the reaction solution was neutralized to about 7 with a hydrochloric acid solution. After neutralization, chloroform (50 g) was added, and the mixture was stirred at room temperature for 15 minutes, and then the organic layer was recovered. The organic layer was concentrated, redissolved in ethyl acetate (100 g), hexane (50 g) was added, and the mixture was stirred at room temperature for 15 minutes to precipitate the product. The precipitate was collected by suction filtration using a 5A filter paper, washed with hexane (50 g), suction filtered using a 5A filter paper, and vacuum dried to obtain compound (p2). Yield 6.8g.
^{_{1 H-NMR (d 6 -DMSO}} ): 2.72ppm (t, 2H, -NH- CH 2 -CH 2 - (O-CH 2 -CH 2) j -O-CH 3) 3.24ppm (s, _{3H, -NH-CH 2 -CH 2} - (O-CH 2 -CH 2) j -O- CH 3), 3.48ppm (m, about _{1,900H, - CH 2 -NH-CH} 2 - CH 2 _{- (O- CH 2 -CH 2)} j -O-CH 3), 5.52ppm (broad, 1H, - NH -CH 2 -COOH)

[Example 1-3]

L-Phenylalanyl-glycine (Fmoc-Phe-Gly) (0.44 g) with N-terminal protected with 9-fluorenylmethyloxycarbonyl group (Fmoc group) and average molecular weight = 20,000, NOF CORPORATION Acetonitrile (40 g) was added and dissolved in "SUNBRIGHT MEPA-20T" (10 g) manufactured by Japan. Then diisopropylethylamine (1.56 g) and 4- (4,6-dimethoxy-1,3,5-triazine-2-yl) -4-methylmorpholinium chloride n hydrate (DMT-MM) (0). .22 g) was added and reacted at room temperature in a nitrogen atmosphere for 1 hour. After completion of the reaction, the mixture was concentrated at 40 ° C., toluene (100 g) was added to the concentrate, the mixture was stirred so as to be uniform, and then suction filtration was performed using a 5A filter paper. Hexane (100 g) was added to the obtained filtrate and stirred at room temperature for 15 minutes to precipitate the product. After suction filtration using 5A filter paper to collect the precipitate, it was dissolved again in toluene (100 g), hexane (100 g) was added, and the mixture was stirred at room temperature for 15 minutes to precipitate the product. The precipitate was collected by suction filtration using a 5A filter paper, washed with hexane (50 g), suction filtered using a 5A filter paper, and vacuum dried to obtain the above compound (p3). Yield 8.6g.
_{^{1 H-NMR (d 6 -DMSO}} ): 1.62ppm (m, 2H, -CO-NH-CH 2 - CH 2 -CH 2 -O- (CH 2 -CH 2 -O) j-CH 3), _{2.80ppm (m, 1H, -NH-} CO-CH- CH 2 -C 6 H 5), 3.04ppm (m, 1H, -NH-CO-CH- CH 2 -C 6 H 5), 3. _{10ppm (m, 2H, -CO-} NH- CH 2 -CH 2 -CH 2 -O- (CH 2 -CH 2 -O) j-CH 3), 3.24ppm (s, 3H, -CO-NH- _{CH 2 -CH 2 -CH 2 -O- (} CH 2 -CH 2 -O) j- CH 3), 3.48ppm (m, about _{1,900H, -CO-NH-CH 2} -CH 2 -CH 2 _{-O- (CH 2 -CH 2 -O)} j-CH 3), 4.20ppm (m, 4H), 7.33ppm (m, 9H), 7.66ppm (m, 4H, Ar), 7.88ppm (D, 2H, Ar), 8.27ppm (t, 1H)

[Example 1-4]

Compound (p3) (8.0 g) was dissolved in acetonitrile (40 g). Then, piperidine (0.86 g) was added, and the mixture was reacted at room temperature in a nitrogen atmosphere for 2 hours. After completion of the reaction, 20% saline solution (80 g) was added, and the mixture was washed by stirring at room temperature for 15 minutes. After separating the organic layer and the aqueous layer, 20% saline (80 g) was added to the organic layer again, and the mixture was washed by stirring at room temperature for 15 minutes to recover the organic layer. The obtained organic layer was concentrated at 40 ° C., toluene (200 g) and magnesium sulfate (10 g) were added to the concentrate, and the mixture was stirred at room temperature for 30 minutes for dehydration and then suction filtered using a 5A filter paper. .. Hexane (100 g) was added to the obtained filtrate and stirred at room temperature for 15 minutes to precipitate the product. The precipitate was collected by suction filtration using a 5A filter paper, washed with hexane (100 g), suction filtered using a 5A filter paper, and vacuum dried to obtain the above compound (p4). Yield 7.2 g.
HPLC: amine purity 92%.
_{^{1 H-NMR (d 6 -DMSO}} ): 1.62ppm (m, 2H, -CO-NH-CH 2 - CH 2 -CH 2 -O- (CH 2 -CH 2 -O) j-CH 3), 1.64ppm (broad, 1H), 2.59ppm (dd, 1H, -NH-CO-CH- CH 2 -C 6 H 5), 2.98ppm (dd, 1H, -NH-CO-CH- CH 2 _{-C 6 H 5), 3.10ppm (} q, 2H, -CO-NH- CH 2 -CH 2 -CH 2 -O- (CH 2 -CH 2 -O) j-CH 3), 3.24ppm ( _{s, 3H, -CO-NH-} CH 2 -CH 2 -CH 2 -O- (CH 2 -CH 2 -O) j- CH 3), 3.48ppm (m, about 1,900H, -CO-NH _{-CH 2 -CH 2 -CH 2 -O- (} CH 2 -CH 2 -O) j-CH 3), 7.24ppm (m, 6H, -NH-CO-CH-CH 2 - C 6 H 5, -NH-), 7.73ppm (t, 1H), 8.12ppm (road, 1H)

[Example 1-5]

The compound (p4) (6.0 g), sodium acetate (49 mg), and glutaric anhydride (51 mg) obtained in Examples 1-4 were dissolved in toluene (25 g) at 40 ° C. for 7 hours in a nitrogen atmosphere. It was reacted. After completion of the reaction, the mixture was diluted with toluene (20 g), suction filtered using a 5A filter paper, hexane (30 g) was added to the obtained filtrate, and the mixture was stirred at room temperature for 15 minutes to precipitate the product. The precipitate was collected by suction filtration using a 5A filter paper. The obtained precipitate was dissolved in ethyl acetate (100 g), hexane (50 g) was added, and the mixture was stirred at room temperature for 15 minutes to precipitate the product. The precipitate was collected by suction filtration using a 5A filter paper, washed with hexane (50 g), suction filtered using a 5A filter paper, and vacuum dried to obtain the above compound (p5). Yield 4.8g.
_{^{1 H-NMR (d 6 -DMSO}} ): 1.62ppm (m, 2H, -CO-NH-CH 2 - CH 2 -CH 2 -O- (CH 2 -CH 2 -O) j-CH 3), _{1.64ppm (broad, 1H), 2.05ppm} (dd, 2H, -NH-CO-CH 2 - CH 2 -CH 2 -COOH) 2.30ppm (m, 4H, -NH-CO- CH 2 -CH _{2 - CH 2 -COOH), 2.59ppm} (dd, 1H, -NH-CO-CH- CH 2 -C 6 H 5), 2.98ppm (dd, 1H, -NH-CO-CH- CH 2 - _{C 6 H 5), 3.10ppm (} q, 2H, -CO-NH- CH 2 -CH 2 -CH 2 -O- (CH 2 -CH 2 -O) j-CH 3), 3.24ppm (s _{, 3H, -CO-NH-CH} 2 -CH 2 -CH 2 -O- (CH 2 -CH 2 -O) j- CH 3), 3.48ppm (m, about 1,900H, -CO-NH- _{CH 2 -CH 2 -CH 2 -O- (} CH 2 -CH 2 -O) j-CH 3), 7.24ppm (m, 6H, -NH-CO-CH-CH 2 - C 6 H 5, - NH-), 7.73 ppm (t, 1H), 8.12 ppm (road, 1H)

[Example 1-6]

The compound (p5) (4.5 g) and N-hydroxysuccinimide (103 mg) obtained in Example 1-5 were dissolved in toluene (25 g). Then, dicyclohexylcarbodiimide (139 mg) was added, and the mixture was reacted at 40 ° C. under a nitrogen atmosphere for 3 hours. After completion of the reaction, toluene (50 g) was added to dilute the mixture, suction filtration was performed using a 5A filter paper, hexane (50 g) was added to the obtained filtrate, and the mixture was stirred at room temperature for 15 minutes to precipitate the product. It was. After suction filtration using 5A filter paper to collect the precipitate, it was dissolved in ethyl acetate (100 g), hexane (50 g) was added, and the mixture was stirred at room temperature for 15 minutes to precipitate the product. The precipitate was collected by suction filtration using a 5A filter paper, washed with hexane (50 g), suction filtered using a 5A filter paper, and vacuum dried to obtain the above compound (p6). Yield 3.5g.
The active ester purity is 98% ( ^{1 1} H-NMR).
_{^{1 H-NMR (d 6 -DMSO}} ): 1.62ppm (m, 2H, -CO-NH-CH 2 - CH 2 -CH 2 -O- (CH 2 -CH 2 -O) j-CH 3), _{1.64ppm (broad, 1H), 2.05ppm} (dd, 2H, -NH-CO-CH 2 - CH 2 -CH 2 -CO -), 2.30ppm (m, 4H, -NH-CO- CH 2 _{-CH 2 - CH 2 -COO -)} , 2.59ppm (dd, 1H, -NH-CO-CH- CH 2 -C 6 H 5), 2.72ppm (s, 4H, -CO- CH 2 -CH _{2 -CO -), 2.98ppm (dd} , 1H, -NH-CO-CH- CH 2 -C 6 H 5), 3.10ppm (q, 2H, -CO-NH- CH 2 -CH 2 -CH _{2 -O- (CH 2 -CH 2 -O} ) j-CH 3), 3.24ppm (s, 3H, -CO-NH-CH 2 -CH 2 -CH 2 -O- (CH 2 -CH 2 - _{O) j- CH 3), 3.48ppm} (m, about _{1,900H, -CO-NH-CH 2} -CH 2 -CH 2 -O- (CH 2 -CH 2 -O) j-CH 3), _{7.24ppm (m, 6H, -NH-} CO-CH-CH 2 - C 6 H 5, -NH -), 7.73ppm (t, 1H), 8.12ppm (broad, 1H)

[Example 1-7]

After dissolving the compound (p2) (3.0 g) obtained in Example 1-2 and the compound (p6) (3.0 g) obtained in Example 1-6 in dichloromethane (60 g), triethylamine (95 mg) ) Was added and reacted at room temperature for 8 hours. After the reaction, 20% saline solution (50 g) was added, the mixture was stirred at room temperature for 15 minutes, the reaction solution was washed, and then the organic layer was recovered. Magnesium sulfate (10 g) was added to the organic layer, the mixture was stirred at room temperature for 15 minutes, and then suction filtered using a 5A filter paper. After concentrating the obtained filtrate, the concentrate was dissolved in ethyl acetate (100 g), hexane (50 g) was added, and the mixture was stirred at room temperature for 15 minutes to precipitate the product. The precipitate was collected by suction filtration using a 5A filter paper, then dissolved again in ethyl acetate (100 g), hexane (50 g) was added, and the mixture was stirred at room temperature for 15 minutes to precipitate the product. After suction filtration using 5A filter paper to collect the precipitate, it is washed with hexane (50 g) containing 2,6-di-tert-butyl-p-cresol (BHT) (10 mg) and 5A filter paper. Was suction-filtered and vacuum-dried to obtain the above compound (p7). Yield 4.2 g.
HPLC: Carboxylic acid purity is 95%.
_{^{1 H-NMR (d 6 -DMSO}} ): 1.62ppm (m, 2H, CO-NH-CH 2 - CH 2 -CH 2 -O- (CH 2 -CH 2 -O) j-CH 3), 1 _{.64ppm (broad, 1H), 2.05ppm} (dd, 2H, -NH-CO-CH 2 - CH 2 -CH 2 -CO -), 2.30ppm (m, 4H, -NH-CO- CH 2 - _{CH 2 - CH 2 -CO -)} , 2.59ppm (dd, 1H, -NH-CO-CH- CH 2 -C 6 H 5), 2.98ppm (dd, 1H, -NH-CO-CH- CH _{2 -C 6 H 5), 3.24ppm} (s, 3H, -NH-CH 2 -CH 2 - (O-CH 2 -CH 2) j-O- CH 3), 3.48ppm (m, about 3 _{, 800H, -CH 2 -NH-CH} 2 -CH 2 - (O- CH 2 -CH 2) j-O-CH 3), 4.61ppm (- CH 2 -COOH), 7.24ppm (m, 6H _{, -NH-CO-CH-CH} 2 - C 6 H 5, -NH -), 7.73ppm (t, 1H), 8.12ppm (broad, 1H))

[Example 2]

The compound (p7) (2.0 g) obtained in Example 1-7 and N-Fmoc-ethylenediamine (32 mg) were dissolved in acetonitrile (5.0 g). Then, diisopropylethylamine (16 mg) and DMT-MM (415 mg) were added, and the mixture was reacted at room temperature in a nitrogen atmosphere for 2 hours. Then, piperidine (107 mg) was added, and the mixture was reacted at room temperature in a nitrogen atmosphere for 2 hours. After completion of the reaction, the mixture was concentrated, the concentrate was redissolved in chloroform (50 g), 20% brine (25 g) was added, and the mixture was washed with stirring at room temperature for 15 minutes to recover the organic layer. Magnesium sulfate (10 g) was added to the obtained organic layer, and the mixture was stirred at room temperature for 30 minutes to dehydrate, and then suction filtered using a 5A filter paper. The obtained filtrate was concentrated at 40 ° C., toluene (100 g) was added to the concentrate to dissolve it, hexane (50 g) was added, and the mixture was stirred at room temperature for 15 minutes to precipitate the product. The precipitate was collected by suction filtration using a 5A filter paper, washed with hexane (30 g) containing 6 mg of BHT, suction filtered using a 5A filter paper, and vacuum dried to obtain the above compound (p8). .. Yield 1.3g.
HPLC: Amine purity is 93%.
_{^{1 H-NMR (d 6 -DMSO}} ): 1.62ppm (m, 2H, CO-NH-CH 2 - CH 2 -CH 2 -O- (CH 2 -CH 2 -O) j-CH 3), 1 _{.64ppm (broad, 1H), 2.05ppm} (dd, 2H, -NH-CO-CH 2 - CH 2 -CH 2 -CO -), 2.30ppm (m, 4H, -NH-CO- CH 2 - _{CH 2 - CH 2 -CO -)} , 2.59ppm (dd, 1H, -NH-CO-CH- CH 2 -C 6 H 5), 2.76ppm (m, 2H, -NH-CH 2 - CH 2 _{-NH 2), 2.98ppm (dd,} 1H, -NH-CO-CH- CH 2 -C 6 H 5), 3.24ppm (s, 3H, -NH-CH 2 -CH 2 - (O-CH _{2 -CH 2) j-O- CH} 3), 3.48ppm (m, about _{3,800H, -CH 2 -NH-CH 2} -CH 2 - (O- CH 2 -CH 2) j-O-CH _{3), 7.24ppm (m, 6H} , -NH-CO-CH-CH 2 - C 6 H 5, -NH -), 7.73ppm (t, 1H), 7.80ppm (broad, 1H), 8 .12ppm (road, 1H)

[Example 3]

The compound (p7) (300 mg) obtained in Example 1-7 was dissolved in acetonitrile (2 g). Then, N-hydroxysuccinimide (6 mg) and dicyclohexylcarbodiimide (6 mg) were added, and the mixture was reacted at 40 ° C. under a nitrogen atmosphere for 3 hours. Then, triethylamine (3 mg) and N- (2-aminoethyl) maleimide hydrochloride (5 mg) were added, and the mixture was reacted at 40 ° C. under a nitrogen atmosphere for 3 hours. After completion of the reaction, the mixture was concentrated, the concentrate was dissolved in ethyl acetate (50 g), hexane (30 g) was added, and the mixture was stirred at room temperature for 15 minutes to precipitate the product. The precipitate was collected by suction filtration using a 5A filter paper, then dissolved again in ethyl acetate (50 g), hexane (30 g) was added, and the mixture was stirred at room temperature for 15 minutes to precipitate the product. After collecting the suction filter paper and the precipitate using 5A filter paper, wash with hexane (30 g) containing BHT (6 mg), suction filter using 5A filter paper, and vacuum dry to obtain the above compound (p9). Obtained. Yield 264 mg. Maleimide purity is 92% ( ^{1 1} H-NMR).
_{^{1 H-NMR (d 6 -DMSO}} ): 1.62ppm (m, 2H, CO-NH-CH 2 - CH 2 -CH 2 -O- (CH 2 -CH 2 -O) j-CH 3), 1 _{.64ppm (broad, 1H), 2.05ppm} (dd, 2H, -NH-CO-CH 2 - CH 2 -CH 2 -CO -), 2.30ppm (m, 4H, -NH-CO- CH 2 - _{CH 2 - CH 2 -CO -)} , 2.59ppm (dd, 1H, -NH-CO-CH- CH 2 -C 6 H 5), 2.66ppm (t, 2H, -NH-CO- CH 2 - _{CH 2 -Maleimide), 2.98ppm (dd} , 1H, -NH-CO-CH- CH 2 -C 6 H 5), 3.24ppm (s, 3H, -NH-CH 2 -CH 2 - (O- _{CH 2 -CH 2) j-O-} CH 3), 3.48ppm (m, about _{3,800H, -CH 2 -NH-CH 2} -CH 2 - (O- CH 2 -CH 2) j-O- CH ₃ ), 4.76 ppm (t, 2H, -NH-CO-CH ₂ - CH _2- Maleimide), 6.98 ppm (s, 2H, -CO-CH-CH- CO-), 7.24 ppm (m) _{, 6H, -NH-CO-CH} -CH 2 - C 6 H 5, -NH -), 7.73ppm (t, 1H), 7.80ppm (broad, 1H), 8.01ppm (broad, 1H), 8.12ppm (road, 1H)

[Example 4]

Toluene (6 g) was added to the compound (p7) (1.2 g) obtained in Example 1-7, and the mixture was heated and dissolved at 40 ° C. Then, N-hydroxysuccinimide (24 mg) and dicyclohexylcarbodiimide (24 mg) were added, and the mixture was reacted at 40 ° C. under a nitrogen atmosphere for 3 hours. After completion of the reaction, suction filtration was performed using a 5A filter paper, the obtained filtrate was diluted with ethyl acetate (100 g), hexane (50 g) was added, and the mixture was stirred at room temperature for 30 minutes to precipitate the product. After suction filtration using a 5A filter paper to collect the precipitate, it was washed with hexane (50 g) containing BHT (10 mg), suction filtered using a 5A filter paper, vacuum dried to obtain the above compound (p10). Obtained. Yield 1.0g. The active ester purity is 91% ( ^{1 1} H-NMR).
_{^{1 H-NMR (d 6 -DMSO}} ): 1.62ppm (m, 2H, CO-NH-CH 2 - CH 2 -CH 2 -O- (CH 2 -CH 2 -O) j-CH 3), 1 _{.64ppm (broad, 1H), 2.05ppm} (dd, 2H, -NH-CO-CH 2 - CH 2 -CH 2 -CO -), 2.30ppm (m, 4H, -NH-CO- CH 2 - _{CH 2 - CH 2 -CO -)} , 2.59ppm (dd, 1H, -NH-CO-CH- CH 2 -C 6 H 5), 2.72ppm (s, 4H, -CO- CH 2 -CH 2 _{-CO -), 2.98ppm (dd,} 1H, -NH-CO-CH- CH 2 -C 6 H 5), 3.24ppm (s, 3H, -NH-CH 2 -CH 2 - (O-CH _{2 -CH 2) j-O- CH} 3), 3.48ppm (m, about _{3,800H, -CH 2 -NH-CH 2} -CH 2 - (O- CH 2 -CH 2) j-O-CH _{3), 4.61ppm (- CH 2} -OCO-succinimide), 7.24ppm (m, 6H, -NH-CO-CH-CH 2 - C 6 H 5, -NH -), 7.73ppm (t, 1H), 8.12ppm (road, 1H)

[Example 5]

[Example 5-1]

The compound (p7) (800 mg) obtained in Example 1-7 was dissolved in toluene (7 g) by heating at 40 ° C., then 3-aminopropionaldehyde diethyl acetal (9 mg) was added, and the temperature was 50 ° C. The reaction was carried out in a nitrogen atmosphere for 2 hours. After completion of the reaction, ethyl acetate (100 g) was added, the mixture was stirred until uniform, hexane (50 g) was added, and the mixture was stirred at room temperature for 15 minutes to precipitate the product. After suction filtration using a 5A filter paper to collect the precipitate, it was washed with hexane (50 g) containing BHT (10 mg), suction filtered using a 5A filter paper, vacuum dried to obtain the above compound (p11). Obtained. Yield 684 mg.
^{_{1 H-NMR (d 6 -DMSO}} ): 1.20ppm (t, 6H (CH 3 -CH 2 -O) 2 -CH-) 1.62ppm (m, 2H, CO-NH-CH 2 - CH 2 - CH _2- O- (CH _{2 -CH 2-} O) j-CH ₃ ), 1.64ppm (road, 1H), 1.85ppm (dd, 2H, -NH-CH ₂ - CH _2- CH- (O) _{-CH 2 -CH 3) 2),} 2.05ppm (dd, 2H, -NH-CO-CH 2 - CH 2 -CH 2 -CO -), 2.30ppm (m, 4H, -NH-CO- CH _{2 -CH 2 - CH 2 -CO -} ), 2.59ppm (dd, 1H, -NH-CO-CH- CH 2 -C 6 H 5), 2.98ppm (dd, 1H, -NH-CO-CH _{- CH 2 -C 6 H 5)} , 3.24ppm (s, 3H, -NH-CH 2 -CH 2 - (O-CH 2 -CH 2) j-O- CH 3), 3.48ppm (m, about _{3,800H, -CH 2 -NH-CH 2} -CH 2 - (O- CH 2 -CH 2) j-O-CH 3), 3.91ppm (- CH 2 -CO-NH-CH 2 -CH _{2 -CH- (O-CH 2 -CH} 3) 2), 4.55ppm (t, 1H, - CH - (O-CH 2 -CH 3) 2) 7.24ppm (m, 6H, -NH-CO _{-CH-CH 2 - C 6 H} 5, -NH -), 7.73ppm (t, 1H), 8.12ppm (broad, 1H)

[Example 5-2]

The compound (p11) (600 mg) obtained in Example 5-1 was dissolved in a phosphate buffer solution (6.0 g) adjusted to pH 1.90, and reacted at room temperature in a nitrogen atmosphere for 3 hours. After the reaction, a 0.1N aqueous sodium hydroxide solution was added to adjust the pH to 6.5, and then sodium chloride (1.5 g) was added and dissolved. A 0.1N aqueous sodium hydroxide solution was added to the obtained solution to adjust the pH to 7.10, then chloroform (10 g) containing BHT (2 mg) was added, and the mixture was stirred at room temperature for 20 minutes to form an organic layer. The product was extracted in. After separating the organic layer and the aqueous layer and recovering the organic layer, chloroform (20 g) containing BHT (4 mg) was added to the aqueous layer again, and the mixture was stirred at room temperature for 20 minutes to extract the product into the organic layer. .. The organic layers obtained in the first and second extractions were combined and concentrated at 40 ° C., the obtained concentrate was dissolved in ethyl acetate (50 g), hexane (30 g) was added, and the mixture was stirred at room temperature for 15 minutes. The product was precipitated. After suction filtration using a 5A filter paper to collect the precipitate, it was washed with hexane (30 g) containing BHT (6 mg), suction filtered using a 5A filter paper, vacuum dried to obtain the above compound (p12). Obtained. Yield 457 mg. Aldehyde purity is 90% ( ^{1 1} H-NMR).
_{^{1 H-NMR (d 6 -DMSO}} ): 1.62ppm (m, 2H, CO-NH-CH 2 - CH 2 -CH 2 -O- (CH 2 -CH 2 -O) j-CH 3), 1 _{.64ppm (broad, 1H), 2.05ppm} (dd, 2H, -NH-CO-CH 2 - CH 2 -CH 2 -CO -), 2.30ppm (m, 4H, -NH-CO- CH 2 - _{CH 2 - CH 2 -CO -)} , 2.59ppm (dd, 1H, -NH-CO-CH- CH 2 -C 6 H 5), 2.66ppm (dd, 2H, -CO-NH-CH 2 - _{CH 2 -CHO), 2.98ppm (dd} , 1H, -NH-CO-CH- CH 2 -C 6 H 5), 3.24ppm (s, 3H, -NH-CH 2 -CH 2 - (O- _{CH 2 -CH 2) j-O-} CH 3), 3.48ppm (m, about _{3,800H, -CH 2 -NH-CH 2} -CH 2 - (O- CH 2 -CH 2) j-O- _{CH 3), 3.91ppm (s,} 2H, - CH 2 -CO-NH-CH 2 -CH 2 -CHO) 7.24ppm (m, 6H, -NH-CO-CH-CH 2 - C 6 H 5 , -NH -), 7.73ppm (t , 1H), 8.12ppm (broad, 1H), 9.72ppm (s, 1H, -CO-NH-CH 2 -CH 2 - CHO)

[Example 6]

[Example 6-1]

L-glutamic acid (Fmoc-Glu-OH) (16.0 mg) whose N-terminal was protected with a 9-fluorenylmethyloxycarbonyl group (Fmoc group) and the compound (p4) (2) obtained in Example 1-4. Acetonitrile (10 g) was added to (0.0 g), and the mixture was heated and dissolved at 30 ° C. Then, diisopropylethylamine (15 mg) and DMT-MM (39.0 mg) were added, and the mixture was reacted at room temperature in a nitrogen atmosphere for 1 hour. Then, piperidine (111 mg) was added, and the mixture was reacted at room temperature in a nitrogen atmosphere for 2 hours. After completion of the reaction, the reaction solution was diluted with toluene (80 g), hexane (40 g) was added, and the mixture was stirred at room temperature for 15 minutes to precipitate the product. After suction filtration using 5A filter paper to collect the precipitate, it was dissolved again in toluene (100 g), hexane (50 g) was added, and the mixture was stirred at room temperature for 15 minutes to precipitate the product. After suction filtration using a 5A filter paper to collect the precipitate, it was washed with hexane (50 g) containing BHT (10 mg), suction filtered using a 5A filter paper, vacuum dried to obtain the above compound (p13). Obtained. Yield 1.6g.
HPLC: amine purity 92%.
¹ H-NMR (d _6- DMSO): 1.54 ppm (m, 2H, -NH-CO-CH (NH ₂ ) -CH _2- CH _2- ), 1.62 ppm (m, 4H, -CO-NH) -CH ₂ - CH _2- CH _2- ), 1.97ppm (m, 2H, -NH-CO-CH (NH ₂ ) -CH ₂ - CH _2- ), 2.74ppm (dd, 1H, -CO-) _{NH-CH- CH 2 -C 6 H} 5), 2.81ppm (dd, 1H, -CO-NH-CH- CH 2 -C 6 H 5), 3.11ppm (m, 11H), 3.24ppm ( _{s, 6H, -CO-NH-} CH 2 -CH 2 -CH 2 -O- (CH 2 -CH 2 -O) j- CH 3), 3.64ppm (m, about 3,800H, -CO-NH _{-CH 2 -CH 2 -CH 2 -O- (} CH 2 -CH 2 -O) j-CH 3), 4.49ppm (m, 1H, -CO-NH- CH -CH 2 -C 6 H 5) _{, 4.57ppm (m, 1H, -CO} -NH- CH -CH 2 -C 6 H 5), 7.25ppm (m, 10H, -CO-NH-CH-CH 2 - C 6 H 5), 7 .74ppm (m, 2H), 8.44ppm (m, 2H), 8.61ppm (m, 2H)

[Example 6-2]

ε-Caprolactone (114 mg) was dissolved in 1N NaOH (0.8 mL) and reacted for 2 hours to prepare a 6-hydroxycaproic acid aqueous solution (0.88 M). Further, the compound (p13) (1.5 g) obtained in Example 6-1 was dissolved in acetonitrile (6 g). Then, the above 6-hydroxycaproic acid aqueous solution (80 μL), diisopropylethylamine (15 mg) and DMT-MM (16 mg) were added, and the mixture was reacted at room temperature in a nitrogen atmosphere for 1 hour. After completion of the reaction, the reaction solution was concentrated at 40 ° C., and chloroform (30 g) was added to the obtained concentrate to dissolve it. A saturated aqueous sodium hydrogen carbonate solution (15 g) was added, and the mixture was washed by stirring at room temperature for 15 minutes. After separating the aqueous layer and the organic layer, a saturated aqueous sodium hydrogen carbonate solution (15 g) was added to the organic layer again, and the mixture was washed by stirring at room temperature for 15 minutes to recover the organic layer. Magnesium sulfate (5 g) was added to the obtained chloroform solution, and the mixture was stirred for 30 minutes to dehydrate, and then suction filtration was performed using a 5A filter paper. The obtained filtrate was concentrated at 40 ° C., ethyl acetate (50 g) was added to the concentrate and stirred to make it uniform, then hexane (25 g) was added, and the mixture was stirred at room temperature for 15 minutes to produce the product. Was precipitated. The precipitate was collected by suction filtration using a 5A filter paper, then dissolved again in ethyl acetate (50 g), hexane (25 g) was added, and the mixture was stirred at room temperature for 15 minutes to precipitate the product. After suction filtration using a 5A filter paper to collect the precipitate, it was washed with hexane (50 g) containing BHT (10 mg), suction filtered using a 5A filter paper, and vacuum dried to obtain the above compound (p14). It was. Yield 1.2g.
^{_{1 H-NMR (CDCl 3)}} : 1.37ppm (m, 2H, HO-CH 2 -CH 2 - CH 2 -CH 2 -CH 2 -CO-NH -), 1.55ppm (m, 4H, HO- _{CH 2 - CH 2 -CH 2 -} CH 2 -CH 2 -CO-NH -), 1.77ppm (m, 4H, -CO-NH-CH 2 - CH 2 -CH 2 -O- (CH 2 -CH _{2 -O) j-CH 3)} , 1.85ppm (m, 1H), 2.01ppm (m, 2H, HO-CH 2 -CH 2 -CH 2 -CH 2 - CH 2 -CO-NH-), 3.01ppm (m, 1H), 3.24ppm (m, 8H), 3.38ppm (s, 6H, -CO-NH-CH 2 -CH 2 -CH 2 -O- (CH 2 -CH 2 -O _{) j- CH 3), 3.64ppm (} m, about _{3,800H, -CO-NH-CH 2} -CH 2 -CH 2 -O- (CH 2 -CH 2 -O) j-CH 3), 4 .03ppm (m, 4H), 4.14ppm (m, 1H), 4.48ppm (m, 2H, -CO-NH- CH -CH 2 -C 6 H 5), 6.95ppm (broad, 1H), 7.00ppm (broad, 1H), 7.26ppm (m, 10H, -CO-NH-CH-CH 2 - C 6 H 5), 7.66ppm (broad, 1H), 8.29ppm (broad, 1H)

[Example 6-3]

The compound (p14) (500 mg) obtained in Example 6-2 was dissolved in dichloromethane (3.5 g). Then, di (N-succinimidyl) (51 mg) and pyridine (20 mg) were added, and the mixture was reacted at room temperature in a nitrogen atmosphere for 8 hours. After completion of the reaction, the reaction solution was washed with 5% saline (5 g), magnesium sulfate (0.1 g) was added, and the mixture was stirred at 25 ° C. for 30 minutes, and then suction filtration was performed using a 5A filter paper. The obtained filtrate was concentrated, ethyl acetate (100 g) was added to the concentrate to dissolve it, hexane (50 g) was added, and the mixture was stirred at room temperature for 15 minutes to precipitate the product. After suction filtration using a 5A filter paper to collect the precipitate, it was washed with hexane (25 g) containing BHT (5 mg), suction filtered using a 5A filter paper, and vacuum dried to obtain the above compound (p15). It was. Yield 286 mg. Active carbonate purity is 91% ( ^{1 1} H-NMR)
_{^{1 H-NMR (CDCl 3)}} : 1.38ppm (m, 2H, Succinimide-OCO-CH 2 -CH 2 - CH 2 -CH 2 -CH 2 -CO-NH -), 1.59ppm (m, 2H, _{succinimide-OCO-CH 2 -CH 2} -CH 2 - CH 2 -CH 2 -CO-NH -), 1.75ppm (m, 6H), 1.85ppm (m, 1H), 2.13ppm (m, 2H _{, succinimide-OCO-CH 2 -CH} 2 -CH 2 -CH 2 - CH 2 -CO-NH -), 2.83ppm (s, 4H, -CO- CH 2 -CH 2 -CO -), 3.01ppm (m, 1H), 3.19ppm ( m, 6H), 3.38ppm (s, 6H, -CO-NH-CH 2 -CH 2 -CH 2 -O- (CH 2 -CH 2 -O) j- CH _3), 3.64ppm (m, about _{3,800H, -CO-NH-CH 2} -CH 2 -CH 2 -O- (CH 2 -CH 2 -O) j-CH 3), 4.03ppm ( m, 3H), 4.18ppm (m , 1H), 4.31ppm (t, 2H, succinimide-OCO- CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CO-NH -), 4.50ppm _{(m, 2H, -CO-NH-} CH -CH 2 -C 6 H 5), 6.98ppm (broad, 1H), 7.15ppm (broad, 1H), 7.26ppm (m, 10H, -CO- _{NH-CH-CH 2 - C} 6 H 5), 7.81ppm (broad, 1H), 8.37ppm (broad, 1H)

[Example 6-4]

After dissolving the compound (p2) (125 mg) obtained in Example 1-2 and the compound (p15) (250 mg) obtained in Example 6-3 in dichloromethane (50 g), triethylamine (0.5 g) was added. It was added and reacted at room temperature for 8 hours. After the reaction, 20% saline solution (20 g) was added, the mixture was stirred at room temperature for 15 minutes, the reaction solution was washed, and then the organic layer was recovered. Magnesium sulfate (5 g) was added to the organic layer, the mixture was stirred at room temperature for 15 minutes, and then suction filtered using a 5A filter paper. After concentrating the obtained filtrate, the concentrate was dissolved in ethyl acetate (100 g), hexane (50 g) was added, and the mixture was stirred at room temperature for 15 minutes to precipitate the product. After suction filtration using a 5A filter paper to collect the precipitate, it was washed with hexane (20 g) containing BHT (4 mg), suction filtered using a 5A filter paper, vacuum dried to obtain the above compound (p16). Obtained. Yield 242 mg.
HPLC: Carboxylic acid purity is 90%
_{^{1 H-NMR (d 6 -DMSO}} ): 1.29ppm (m, 2H, -NH-CO-CH 2 -CH 2 - CH 2 -CH 2 -CH 2 -OCO -), 1.58ppm (m, 4H _{, -NH-CO-CH 2 -} CH 2 -CH 2 - CH 2 -CH 2 -OCO -), 1.75ppm (m, 6H), 1.85ppm (m, 1H), 2.13ppm (m, 2H _{, -NH-CO- CH 2 -CH 2} -CH 2 -CH 2 -CH 2 -OCO -), 3.01ppm (m, 1H), 3.19ppm (m, 6H), 3.38ppm (s, 6H _{, -CO-NH-CH 2 -CH} 2 -CH 2 -O- (CH 2 -CH 2 -O) j- CH 3), 3.64ppm (m, about 5,700H, -CO-NH-CH _{2 -CH 2 -CH 2 -O- (CH 2} -CH 2 -O) j-CH 3), 3.90ppm (t, 2H, -NH-CO-CH 2 -CH 2 -CH 2 -CH 2 - CH _2- OCO-) 4.03 ppm (m, 3H) 4.18 ppm (m, 1H) 4.37 ppm (s, 2H, -CH _2- COOH) 4.50 ppm (m, 2H, -CO-NH-) _{CH -CH 2 -C 6 H 5)} , 6.98ppm (broad, 1H), 7.15ppm (broad, 1H), 7.26ppm (m, 10H, -CO-NH-CH-CH 2 - C 6 H ₅ ), 7.81ppm (road, 1H), 8.37ppm (road, 1H)

[Comparative Example 1]

[Comparative Example 1-1]

After dissolving the compound (1 g) obtained in Example 1-2 and "SUNBRIGHT ME-200GS3" (1 g) manufactured by NOF CORPORATION having an average molecular weight of 20,000 in dichloromethane (20 g), triethylamine (0. 2 g) was added and reacted at room temperature for 8 hours. After the reaction, 20% saline solution (10 g) was added, the mixture was stirred at room temperature for 15 minutes, the reaction solution was washed, and then the organic layer was recovered. Magnesium sulfate (3 g) was added to the organic layer, the mixture was stirred at room temperature for 15 minutes, and then suction filtered using a 5A filter paper. After concentrating the obtained filtrate, the concentrate was dissolved in ethyl acetate (100 g), hexane (50 g) was added, and the mixture was stirred at room temperature for 15 minutes to precipitate the product. After suction filtration using a 5A filter paper to collect the precipitate, it was washed with hexane (30 g) containing BHT (6 mg), suction filtered using a 5A filter paper, and vacuum dried to obtain the above compound (p17). Obtained. Yield 1.2g.
HPLC: Carboxylic acid purity is 93%.
_{^{1 H-NMR (d 6 -DMSO}} ): 1.73ppm (m, 2H, -CO-NH-CH 2 - CH 2 -CH 2 - (O-CH 2 -CH 2) j -), 2.03ppm ( _{m, 2H, -NH-CO-} CH 2 - CH 2 -CH 2 -CO-N -), 2.34ppm (t, 4H, -NH-CO- CH 2 -CH 2 - CH 2 -CO-N- _{), 3.40ppm (s, 6H,} - (O-CH 2 -CH 2) j-O- CH 3), 3.55ppm (m, about _{3,800H, -CH 2 - (O- CH} 2 -CH _{2) j-O-CH 3} ), 4.61ppm (s, 2H, - CH 2 -COOH), 7.70ppm (broad, 1H, -CH 2 - NH -CO-CH 2 -CH 2 -CH 2 - CO-)

[Comparative Example 1-2]

The compound (p17) (1.0 g) obtained in Comparative Example 1-1 and N-Fmoc-ethylenediamine (16 mg) were dissolved in acetonitrile (2.5 g). Then, diisopropylethylamine (8 mg) and DMT-MM (208 mg) were added, and the mixture was reacted at room temperature in a nitrogen atmosphere for 2 hours. Then, piperidine (107 mg) was added, and the mixture was reacted at room temperature in a nitrogen atmosphere for 2 hours. After completion of the reaction, the mixture was concentrated, the concentrate was redissolved in chloroform (50 g), 20% brine (25 g) was added, and the mixture was washed with stirring at room temperature for 15 minutes to recover the organic layer. Magnesium sulfate (10 g) was added to the obtained organic layer, and the mixture was stirred at room temperature for 30 minutes to dehydrate, and then suction filtered using a 5A filter paper. The obtained filtrate was concentrated at 40 ° C., toluene (100 g) was added to the concentrate to dissolve it, hexane (50 g) was added, and the mixture was stirred at room temperature for 15 minutes to precipitate the product. The precipitate was collected by suction filtration using a 5A filter paper, washed with hexane (30 g) containing 6 mg of BHT, suction filtered using a 5A filter paper, and vacuum dried to obtain the above compound (p8). .. Yield 581 mg.
HPLC: Amine purity is 90%.
_{^{1 H-NMR (d 6 -DMSO}} ): 1.73ppm (m, 2H, -CO-NH-CH 2 - CH 2 -CH 2 - (O-CH 2 -CH 2) j -), 2.03ppm ( _{m, 2H, -NH-CO-} CH 2 - CH 2 -CH 2 -CO-N -), 2.34ppm (t, 4H, -NH-CO- CH 2 -CH 2 - CH 2 -CO-N- _{), 2.76ppm (m, 2H,} -CH 2 -CO-NH-CH 2 - CH 2 -NH 2), 3.40ppm (s, 6H, - (O-CH 2 -CH 2) j-O- CH _3), 3.55ppm (m, about _{3,800H, -CH 2 - (O- CH} 2 -CH 2) j-O-CH 3), 3.66ppm (m, 2H, -CH 2 -CO- _{NH- CH 2 -CH 2 -NH 2)} , 3.91ppm (s, 2H, -N- CH 2 -CO-NH -), 7.70ppm (broad, 1H, -CH 2 - NH -CO-CH 2 _{-CH 2 -CH 2 -CO -),} 7.83ppm (broad, 1H, -CH 2 -CO- NH -CH 2 -CH 2 -NH 2)

[Example 8]
Stability test in serum 1 mL of mouse or human serum was added to a 1.5 mL Eppendorf tube, and the compound (p8) which is a branched degradable polyethylene glycol derivative obtained in Example 2 was used, Comparative Example 1-2. The compound (p18), which is a non-degradable polyethylene glycol derivative obtained in the above, and methoxyPEGamine 40 kDa were added at a concentration of 5.0 mg / mL, respectively. After incubation at 37 ° C. for 96 hours, 200 μL was sampled, acetonitrile was added thereto, and the mixture was stirred with vortex for 1 minute to precipitate proteins in serum, centrifuged, and the supernatant was recovered. Next, in order to remove hydrophobic substances such as fatty acids, hexane was added to the recovery liquid, the mixture was stirred with a vortex for 1 minute, centrifuged, and the lower layer was recovered. This solution was concentrated under vacuum conditions to recover the polyethylene glycol derivative from the serum. Then, GPC analysis was performed to calculate the decomposition rate of the degradable polyethylene glycol derivative.
The decomposition rate was calculated by the following formula.
Decomposition rate = (40 kDa peak area% before test − 40 kDa peak area% after test) ÷ (40 kDa peak area% before test) × 100
The results are shown in Table 1.

According to Table 1, the compound (p8) which is a branched degradable polyethylene glycol derivative is decomposed in serum like the compound (p18) which is a non-degradable polyethylene glycol derivative and methoxyPEGamine 40 kDa. I couldn't. That is, it was shown that the degradable polyethylene glycol derivative is stable in blood.

[Example 9]
Degradability test using cells Using 10 mL of medium RPMI-1640 (10% FBS Pn / St), RAW264.7 was seeded on a 100 mm dish in 10 × 106 cells, cultured at 37 ° C. for 24 hours, and then obtained in Example 2. The compound (p8) which is a branched degradable polyethylene glycol derivative, the compound (p18) which is a non-degradable polyethylene glycol derivative obtained in Comparative Example 1-2, and methoxyPEGamine 40 kDa at a concentration of 10 mg / mL. Each was replaced with a medium dissolved so as to be, and cultured at 37 ° C. for 96 hours. After culturing, the cells are dissolved in 1% SDS solution, diluted with PBS, acetonitrile is added thereto, and the mixture is stirred with vortex for 1 minute to precipitate proteins in the cell lysate, centrifuged, and then above. Qing was recovered. Next, in order to remove hydrophobic substances such as fatty acids, hexane was added to the recovery liquid, the mixture was stirred with a vortex for 1 minute, centrifuged, and the lower layer was recovered. This solution was concentrated under vacuum conditions to recover the polyethylene glycol derivative from the cells.
In addition, in order to confirm the decomposition in the medium used for cell culture, various polyethylene glycol derivatives were cultured only in the medium dissolved at a concentration of 10 mg / mL at 37 ° C. for 96 hours, and polyethylene was cultured in the same operation as above. The glycol derivative was recovered.
Then, GPC analysis of various recovered polyethylene glycol derivatives was performed, and the decomposition rate of the branched degradable polyethylene glycol derivative was calculated by the same formula as in Example 8.
The results are shown in Table 2.

According to Table 2, it was confirmed that the compound (p8), which is a branched degradable polyethylene glycol derivative, is effectively decomposed intracellularly (decomposition rate 99%) and decomposes to a molecular weight of 40,000 to 20,000. did it. Since the branched degradable polyethylene glycol derivative did not decompose in the medium used for cell culture, it was confirmed that it was specifically decomposed inside the cells. On the other hand, neither the compound (p18), which is a non-degradable polyethylene glycol derivative, nor the methoxyPEGamine 40 kDa was intracellularly degraded.

The branched degradable polyethylene glycol derivative of the present invention is a high molecular weight polyethylene glycol derivative that does not cause cell vacuoles, and can be effectively used for modifying biological substances, and can be effectively used in blood in a living body. It is stable and is degraded intracellularly.

Claims (12)

A branched degradable polyethylene glycol derivative represented by the following formula (1), which has an oligopeptide that decomposes intracellularly in the molecule.

(In the formula, k ₁ and k ₂ are independently 1 to 12, j ₁ and j ₂ are independently 45 to 950, respectively, R is a hydrogen atom, and the number of carbon atoms is 1 which is substituted or not substituted. ~ 12 alkyl groups, substituted aryl groups, aralkyl groups or heteroalkyl groups, Z is an oligopeptide that is degraded by intracellular enzymes, X is a functional group that can react with biorelated substances, L ₁ and L ₂ is an independent single bond or divalent spacer.) The branched degradable polyethylene glycol derivative according to claim 1, wherein the oligopeptide of Z is a 2 to 8 residue degradable oligopeptide composed of a neutral amino acid excluding cysteine. The branched degradable polyethylene glycol derivative according to any one of claims 1 to 2, wherein the oligopeptide of Z is a peptide having glycine as a C-terminal amino acid. The branched degradable polyethylene glycol derivative according to any one of claims 1 to 3, wherein the oligopeptide of Z is an oligopeptide having at least one hydrophobic amino acid having a hydropathy index of 2.5 or more. Claim that L ₁ and L ₂ are independently single bonds, urethane bonds, amide bonds, ether bonds, thioether bonds, secondary amino groups, urea bonds, or alkylene groups containing these bonds and / or groups. The branched degradable polyethylene glycol derivative according to any one of 1 to 4. X is an active ester group, active carbonate group, aldehyde group, isocyanate group, isothiocyanate group, epoxy group, maleimide group, vinylsulfonyl group, acrylic group, sulfonyloxy group, carboxy group, thiol group, dithiopyridyl group, α-haloacetyl The branched degradable polyethylene glycol according to any one of claims 1 to 5, which is selected from the group consisting of a group, an alkynyl group, an allyl group, a vinyl group, an amino group, an oxyamino group, a hydrazide group and an azide group. Derivative. A branched degradable polyethylene glycol derivative represented by the following formula (2).

(In the formula, k ₁ and k ₂ are independently 1 to 12, j ₁ and j ₂ are independently 45 to 950, respectively, R is a hydrogen atom, and the number of carbon atoms is 1 which is substituted or not substituted. It is an alkyl group of ~ 4, a substituted aryl group, an aralkyl group or a heteroalkyl group, W is an oligopeptide of 5 to 47 residues having a symmetrical structure centered on glutamate or lysine, and a is 2 to 8. X is a functional group capable of reacting with a bio-related substance, and L ₁ and L ₂ are independently single-bonded or divalent spacers, respectively.) The branched degradable polyethylene glycol derivative according to claim 7, wherein the oligopeptide having a symmetrical structure centered on glutamic acid or lysine of W is an oligopeptide having the following w1 or w2 structure.

(In the formula, Q is a residue of glutamic acid or lysine, and Z is a degradable oligopeptide of 2 to 5 residues consisting of neutral amino acids excluding cysteine.) The branched degradable polyethylene glycol derivative according to claim 8, wherein the degradable oligopeptide of Z is an oligopeptide having glycine as a C-terminal amino acid. The branched degradability according to any one of claims 8 to 9, wherein the degradable oligopeptide of Z is an oligopeptide having at least one hydrophobic neutral amino acid having a hydropathy index of 2.5 or more. Polyethylene glycol derivative. L ₁ and L ₂ are independent, single bond, urethane bond, amide bond, ether bond, thioether bond, secondary amino group, urea bond, or alkylene group which may contain these bonds and / or groups. The branched degradable polyethylene glycol derivative according to any one of claims 7 to 10. X is an active ester group, active carbonate group, aldehyde group, isocyanate group, isothiocyanate group, epoxy group, maleimide group, vinylsulfonyl group, acrylic group, sulfonyloxy group, carboxy group, thiol group, dithiopyridyl group, α-haloacetyl The branched degradable polyethylene glycol according to any one of claims 7 to 11, selected from the group consisting of a group, an alkynyl group, an allyl group, a vinyl group, an amino group, an oxyamino group, a hydrazide group and an azide group. Derivative.